The present invention relates to an enzyme useful as an oral therapeutic agent and more particularly to a method of producing .alpha.-1, 3-glucan 3-glucanohydrolase which can be used to attack glucans of dental plaque.
Dental plaque is a deposit which accumulates on the teeth and adjacent surfaces in the oral cavity. The plaque is a product of microbial growth resulting from food residues in the mouth. Mucoproteins and minerals present from the saliva and dead cells in the mouth also assist in plaque formation.
Plaque is removed to some extent by effective brushing of the teeth, but the less accessible and more sheltered areas of the mouth which cannot be readily reached by a toothbrush are particularly susceptible to plaque and, eventually, calculus growth. Left unhindered, the plaque increases in size and more tenaciously adheres to the teeth. The bacterial metabolism within the plaque on the tooth surface results in the production of acids, toxins and enzymes which are deleterious to the neighboring oral tissues. There is much evidence pointing to certain bacterial plaques as being the direct cause of dental caries, due to the generation of acids within the plaque structure. In any event plaque is unhealthy and therefore undesirable.
During recent years, it has been found that the organisms often associated with the formation of dental caries are certain oral streptococci belonging to the Streptococcus mutans group which contribute to the build-up of plaque and have been implicated in the development of dental caries and periodontal diseases. Specific strains of Streptococcus mutans can synthesize adhesive glucans which facilitate adherence of the organisms to tooth surfaces and contribute to the build-up of plaque. The significance of such glucans in relation to dental disease has been demonstrated through various investigations with "glucan synthesis-defective" mutants which have exhibited only low levels of virulence in animal experiments. Other streptococcal strains are additionally thought to synthesize fructan (levan) polysaccharides. Still other Actinomyces strains are major constituents of dental plaque and can be related to both root surface caries and periodontal disease. Such organisms also contribute to the formation of plaque through the synthesis of slimy, extracellular and cell-surface polysaccharides.
The extracellular streptococcal glucans are composed of glucosyl units linked in .alpha.-1,3, and .alpha.-1,6 configurations. Past attempts to control dental plaque formation with plaque-dispersing glucanohydrolases such as dextranase which hydrolyze 1,6-glucosyl linkages have had only limited success possibly because the dextranase enzymes only partially degrade the glucans while leaving a water-insoluble residue. Recent investigations, in fact, have shown that this residual material is composed primarily of 1,3-linked glycosyl units with relatively few 1,6 linkages. While sources of 1,3 glucanohydrolases have been reported, to our knowledge the enzymes are not commercially available for oral therapeutic use.
Various other attempts have been made to control cariogenic bacteria and the formation of plaque on teeth. For example, fluoride solutions or gels have been used. Treatment with these materials is typically performed in a dental office at periodic, but not frequent, intervals. The primary objective of these treatments is to render the tooth enamel more resistant to the acid action caused by plaque. Such treatments do not, however, result in plaque control for an extended period since plaque reestablishes itself on the teeth shortly after ingestion of food.
Even when the frequency of application of such solutions and gels is increased only partial control has been shown. For example, studies wherein a fluoride-containing solution (1% fluoride concentration) was applied four to five times in the course of a year have demonstrated that this technique had only limited success due to the rapid reestablishment of plaque in the oral cavity. Moreover, the daily application of a fluoride gel by means of a custom-fitted polyvinyl mouthpiece for a period of twenty-one months also showed no substantial change in plaque formation among treated and untreated patients. See "Clinical Anticaries Effect of Repeated Topical Sodium Fluoride Application By Mouthpiece", Journal of the American Dental Association, V. 75, No. 3, September 1967, pp. 638-644.
Other attempts at inhibiting the formation of plaque have also been made. For example, U.S. Pat. No. 3,733,399 describes toothpaste compositions which contain the enzyme invertase as the active ingredient. Another approach is disclosed in U.S. Pat. No. 3,894,147 wherein the application to teeth of a dialkyl pyrophosphate having from about 8 to 14 carbon atoms in the alkyl groups is described as useful in inhibiting plaque formation.